1. Field of the Invention
This invention relates to the field of biochemical assays and reagents. More specifically, this invention relates to modified fluorescent proteins and to methods for their use.
2. Description of the Related Art
Because of its easily detectable green fluorescence, green fluorescent protein (GFP) from the jellyfish Aequorea Victoria has been used widely to study gene expression and protein localization. GFP fluorescence does not require a substrate or cofactor; hence, it is possible to use this reporter in numerous species and in a wide variety of cells. GFP is a very stable protein which can accumulate and thus is often toxic to mammalian cells.
Recently, crystallographic structures of wild-type GFP and the mutant GFP S65T reveal that the GFP tertiary structure resembles a barrel (Ormo et al. (1996) Science 273:1392-1395;. Yang, F., Moss, L. G., and Phillips, G. N., Jr. (1996) Nature Biotech 14: 1246-1251). The barrel consists of beta sheets in a compact antiparallel structure. In the center of the barrel, an alpha helix containing the chromophore is shielded by the barrel. The compact structure makes GFP very stable under diverse and/or harsh conditions, such as protease treatment, making GFP an extremely useful reporter in general. On the other hand, its stability makes it difficult to determine short-term or repetitive events.
A great deal of research is being performed to improve the properties of GFP and to produce GFP reagents useful for a variety of research purposes. New versions of GFP have been developed via mutation, including a “humanized” GFP DNA, the protein product of which has increased synthesis in mammalian cells (see Cormack, et al., (1996) Gene 173, 33-38; Haas, et al., (1996) Current Biology 6, 315-324; and Yang, et al., (1996) Nucleic Acids Research 24, 4592-4593). One such humanized protein is “enhanced green fluorescent protein” (EGFP). Other mutations to GFP have resulted in blue-, cyan- and yellow-fluorescent light emitting versions.
Ornithine decarboxylase (ODC) is an enzyme critical in the biosynthesis of polyamines. Murine ornithine decarboxylase is one of most short-lived proteins in mammalian cells, with a half life of about 30 minutes (Ghoda, et al., (1989) Science 243, 1493-1495; and Ghoda, et al. (1992) Mol. Cell. Biol. 12, 2178-2185). Rapid degradation of murine ornithine decarboxylase has been determined to be due to the unique composition of its C-terminus, a portion of which has a PEST sequence—a sequence which has been proposed as characterizing short-lived proteins. The PEST sequence contains a region enriched with proline (P), glutamic acid (E), serine (S), and threonine (T), often flanked by basic amino acids, lysine, arginine, or histidine (Rogers, et al., (1989) Science 234:364-68; Reichsteiner, M. (1990) Seminars in Cell Biology 1:433-40).
The ornithine decarboxylase of Trypanosoma brucei (TbODC) does not have a PEST sequence, and is long-lived and quite stable when it is expressed in mammalian cells (Ghoda, et al., (1990) J. Biol. Chem. 265: 11823-11826); however appending the C terminus of murine ornithine decarboxylase to TbODC makes TbODC become unstable. Moreover, deletion of the C-terminal, PEST-containing region from murine ornithine decarboxylase prevents its rapid degradation (Ghoda, L., et al. (1989) Science 243: 1493-1495).
The prior art is deficient in a destabilized or short-lived GFP. The present invention fulfills this need in the art.